Forced sustained swimming exercise at optimal speed enhances growth in many fish species, particularly through hypertrophy of the white skeletal muscle. The exact mechanism of this effect has not been resolved yet. To explore the role of cortisol, we first subjected wild-type zebrafish to an exercise protocol validated for exercise-enhanced growth, and showed that exercised zebrafish, which indeed showed enhanced growth, had higher cortisol levels than the non-exercised controls. A central role was therefore hypothesized for the steroid hormone cortisol acting through the Glucocorticoid receptor (Gr). Second, we subjected wild-type zebrafish and zebrafish with a mutant Gr to exercise at optimal, suboptimal, and super-optimal speeds and compared them with non-exercised controls. Exercised zebrafish showed growth enhancement at all speeds, with highest growth at optimal speeds. In the Gr mutant fish, exercise resulted in growth enhancement similar to wild-type zebrafish, indicating that cortisol signaling through Gr cannot be considered as a main determinant of exercise-enhanced growth. Finally, the transcriptome of white skeletal muscle tissue was analyzed by RNA sequencing. The results of this analysis showed that in the muscle tissue of Gr mutant fish a lower number of genes is regulated by exercise than in wild-type fish (183 vs. 351). A cluster of 36 genes was regulated by exercise in both wild-type and mutant fish, and in this cluster genes involved in transcriptional regulation and protein ubiquitination were overrepresented. Because these two processes appear to be regulated in both wild type and mutant fish, which both display exercise-enhanced growth, we suggest that they play an important role in the growth of muscles upon exercise. Danio rerio . Upon activation by cortisol, the Gr acts as a transcription factor, regulating the expression of a wide variety of target genes. The zebrafish Gr is encoded by a single gene  and one To test this hypothesis, we have investigated in the present study whether exercise-enhanced growth originates from altered cortisol levels. First, the effects of sustained exercise on plasma cortisol levels were assessed. Second, we investigated the effect of cortisol signaling through Gr on exercise-enhanced growth at suboptimal, optimal, and super optimal swimming speeds. For this purpose, an available Gr mutant zebrafish line  was usedThe experimental protocols complied with the current laws of the Netherlands and were approved by the animal experimental committee .D. rerio) of 3 months old were provided by the zebrafish facilities of the Institute of Biology Leiden . A Gr mutant fish line . Water quality was continuously monitored and controlled.Fish were housed for 2 weeks in aquaria with fresh water at 28°C at a photoperiod regime of 16L:8D and fed -1 or ∼10 BL s-1 , 0.4 m s-1 or ∼13 BL s-1  or 0.5 m s-1 or ∼16 BL s-1 , for 6 h day-1 (09.00–15.00 h), for 5 days week-1 and for 4 weeks long. The non-exercise group remained at 0.1 m s-1.After 2 weeks in aquaria, fish were either sampled as start group or divided into an exercise and non-exercise group (group sizes shown below). The exercise and non-exercise groups were introduced in Blazka-type 127 L swim tunnels as described by -1) to the experimental swimming speeds. After swimming for 6 h, the speed was decreased slowly until the resting flow rate of 0.1 m s-1 was reached again. At the minimal flow rate of 0.1 m s-1 all fish could move freely in all directions, while at the flow rate of 0.4 m s-1 fish were forced to swim against the flow at their optimal speed. No mortality occurred among non-exercised and exercised fish due to experimental treatment.After three days of acclimatization in the swim tunnels, the training protocol was started. The motor speed of the tunnels with fish of the exercise group was increased slowly from resting conditions  and measured for body weight (BW) and body length .N = 25) or divided into an exercise and non-exercise group (each N = 25). The exercise group was forced to sustained exercise at an optimal swimming speed of 0.4 m s-1.Two experiments were performed, and an overview of the design of these experiments is presented in Figure N = 10 Gr mutant and N = 10 wild-type fish) or divided into three exercise groups and a non-exercise group (each N = 15 Gr mutant and N = 15 wild-type zebrafish). The three exercise groups were subjected to exercise at sub-optimal swimming speed (0.3 m s-1 or ∼10 BL s-1), optimal swimming speed (0.4 m s-1 or ∼13 BL s-1) or super-optimal swimming speed (0.5 m s-1 or ∼16 BL s-1), while the non-exercise group remained at 0.1 m s-1.In experiment 2, both Gr mutant and wild-type zebrafish were either measured as start group , according to the manufacturer’s instructions.To determine plasma cortisol concentrations, the tails of the fish in experiment 1 were cut and blood was extracted with heparinized micro capillaries. Micro capillaries were then spun for 5 min at 11,000 rpm in a micro centrifuge. Sufficient blood could only be collected for larger fish: After the 4 weeks experimental period in experiment 2, the white muscle fillet was dissected dorsally from the lateral line in the epaxial quadrant along the whole length of the fish and stored in RNAlater (Ambion) at –20°C. Tissue was lysed in QIAzol Lysis Reagent. A Qiagen TissueRuptor was used to cut up the tissue samples and RNA was extracted using the Qiagen miRNeasy Mini Kit according to the manufacturer’s description . RNA was eluted in 50 μl and quantified by Nanodrop . Integrity of the RNA was confirmed using an Agilent Bioanalyzer 2100 total RNA Nanoseries II chip .TM cDNA Synthesis Kit (Bio-Rad). The cDNA samples were used for genotyping using a custom-designed Kompetitive Allele Specific PCR (KASP) genotyping assay. Finally, N = 48 Gr mutant  and N = 45 wild-type  zebrafish were identified.Fish were genotyped after experiment 2 so the SL and BW data could be assigned as belonging to Gr mutant or wild-type zebrafish. Extracted total RNA was reverse-transcribed using the iScriptN = 3) or swam at Uopt (N = 3) for each of both Gr mutant and wild-type zebrafish groups to make all cross comparisons possible. Illumina RNAseq libraries were prepared from 1 μg total RNA using the Illumina TruSeq RNA Sample Prep Kit v2 according to the manufacturer’s instructions . All RNAseq libraries (150–750 bp inserts) were sequenced on an Illumina HiSeq2500 sequencer as 1 × 50 nucleotides single-end reads according to the manufacturer’s protocol. Image analysis and base calling were done using the Illumina pipeline. Total yield varied from ∼9 to ∼16 M reads per sample.RNAseq was performed on RNA of fish that rested  using TopHat  were tested similarly. Responses to exercise were determined for each treatment vs. the controls at the start within each group, and then pairwise compared between groups with Student t-tests. Differences at P ≤ 0.05 were considered significant. Data are presented as averages ± standard errors (AV ± SE).Statistical analyses were carried out with IBM SPSS Statistics 22.0. Datasets were first tested for normal distribution with Kolmogorov–Smirnov tests and all data were normally distributed. As for experiment I, one-way ANOVA was performed with Tukey P = 0.007), weight (P = 0.001) and cortisol levels (P = 0.009). Exercised fish were significantly longer than fish at the start of the experiment . They were 5.6% longer than non-exercised fish but this difference was not significant .In the first experiment, the effects of exercise at optimal swimming speed were tested on size and cortisol levels Figure . Exercis-1, respectively) but due to high individual variation this difference was not significant. Cortisol concentrations of exercised fish were significantly different (P = 0.007) from cortisol data of fish of the start group (0.011 ± 0.003 μg ml-1).The average cortisol levels were 3.5-fold higher in exercised fish vs. non-exercised fish . The growth patterns indicated most pronounced growth when swimming at Uopt (P < 0.01). Gr mutants swimming at Uopt were on average 0.12 g  heavier than the controls at the start and 0.09 g  heavier than the non-exercised fish. Despite the fact that Gr mutants are bigger than wt fish, comparison of treatment effects between both groups showed that groups similarly responded with growth to exercise. Only Gr mutants swimming at super optimal speed showed a significantly different response in weight than the wild type fish that were swimming at this speed (P = 0.008).In the second experiment, the effects of exercise at suboptimal, optimal, and super optimal swimming speed were tested on size of Gr mutant and wild-type zebrafish Figure . In geneP < 0.05 (1.10%). Of these 351 differentially expressed genes (DEGs), expression of 192 genes was upregulated and expression of 159 genes was downregulated by exercising. In Gr mutants, this comparison showed significant differential expression of 183 genes at P < 0.05 (0.57%). Of these 183 DEGs, expression of 49 genes was upregulated and expression of 134 genes was downregulated upon exercise. Comparing exercised and non-exercised fish, the wild-type fish and the Gr mutants have only 36 DEGs in common  fish were used, yielding four experimental groups. In wild type fish, the comparison exercised vs. non-exercised showed significant differential expression of 351 genes at n Figure . Of thesn Figure  and the n Figure . Gene onGene ontology for the 315 specific DEGs for wild-type zebrafish revealed 12 annotation clusters with enrichment scores > 1, with DEGs involved in the zona pellucida; steroid biosynthesis; oxygen transport, heme, and iron; collagen, and others. Gene ontology for the 147 specific DEGs for Gr mutant zebrafish revealed three annotation clusters with enrichment scores > 1, with DEGs involved in hydrolase activity; GTP binding; transport, transporter activity and transmembrane transport.oxidative stress induced growth inhibitor 1, downregulated in both groups. By far most of these genes were, however, differentially regulated when comparing Gr mutant and wild-type zebrafish: genes which were up or down regulated in one group were not significantly regulated in the other group. The most extreme examples are the expressions of growth arrest-specific 7a (fc 12.26) and opioid growth factor receptor-like 1 (fc 9.28) which were upregulated in wild-type zebrafish, and insulin-like growth factor binding protein 6a (fc 40.26) and growth regulation by estrogen in breast cancer 1 (fc 10.61) which were upregulated in Gr mutant fish. Furthermore, expression of several myosin related genes is upregulated in wild-type zebrafish but not in Gr mutant zebrafish. Also nine collagen types were upregulated in wild-type zebrafish but expression of one other type was upregulated in mutants. Finally, it is worth mentioning that the progesterone receptor was upregulated at fc 13.68 in Gr mutant zebrafish while expression was not significantly differential in wild-type zebrafish.Thus, after comparing exercised and non-exercised fish, 351 DEGS were found in the wild-type fish and 183 DEGs in the Gr mutants. When focusing on DEGs involved in muscle growth and structure , occurs. The lowest COT can be calculated by equaling the first derivative of the polynomial function of COT vs. speed to zero . Hereby Exercise-enhanced growth is accompanied by higher cortisol levels in zebrafish than occurring at resting conditions. These results are in contrast with results in other fish species like rainbow trout  and Atlaper se. Cortisol signaling through Gr can therefore not be considered as a main determinant of exercise-enhanced growth. The conclusion is also supported by the fact that salmonids show exercise-enhanced growth in combination with decreased cortisol levels , key regulator in the PI3K/AKT/mTOR pathway toward protein biosynthesis in the muscle, was high but not differentially regulated in or between exercised Gr mutant and wild-type zebrafish. Neither were the expressions of myogenic transcription factors such as paired box proteinpax7 and myocyte enhancer factormef2 differentially regulated, nor the expression of any of the subunits for AMP-activated protein kinase  and peroxisome proliferator-activated receptor gamma coactivator 1-alpha . Therefore we cannot confirm the exercise-induced activation of the PI3K/AKT/mTOR, the mTOR-MEF2 and the AMPK-PGC1α signaling pathways toward muscle growth and proliferation, myogenesis and energy homeostasis, not in wild-type fish, nor in Gr mutants. As Remarkably, expressions of several genes which were considered as key marker genes in a recent study on exercised zebrafish by growth arrest-specific 7a (gas7a) and opioid growth factor receptor-like 1 (ogfrl1), while on the other hand the expressions of insulin-like growth factor binding protein 6a (igfbp6a) and growth regulation by estrogen in breast cancer 1 (greb1) were upregulated in Gr mutants. Both Growth arrest-specific 7a as the Opioid growth factor receptor-like 1 appear to have suppressive effects on growth. Expression of growth arrest-specific 7a occurs in cells that enter a quiescent state  promotes growth by estrogen-dependent proliferation of (breast cancer) cells  and had many specific DEGs . Among these specific DEGs were genes encoding growth factors, proteins involved in muscle contraction  and in the extracellular matrix . Most pronounced differences when comparing genes which were differentially expressed between wild-type zebrafish and Gr mutants, were on the one hand the upregulated expressions of several genes in wild-type zebrafish such as nt state  and may nt state . The Opig growth . Insulinr) cells . Besidesr) cells  which biOther signaling pathways, such as cortisol signaling through the mineralocorticoid receptor  and actiIn conclusion, cortisol signaling cannot be considered as a main mechanism behind exercise-enhanced growth. Moreover, as Gr mutants could fulfil the long term exercise protocol, even at the highest speeds, cortisol signaling cannot even be considered as crucial in the functioning of the white muscle under exercise. However, we demonstrate that cortisol has large effects on the transcriptional response of the muscle tissue to exercise, suggesting that the physiology of the muscle is altered. Finally, we identified several Gr-independent transcriptional responses to exercise, which may be at the base of exercise-enhanced growth.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.AP and MS conceived and designed the study. AP, SM, RD, and MS acquired the data. AP, RD, and MS analyzed and interpreted the data and drafted and reviewed the manuscript. All authors read and approved the final manuscript.RD was employed by company ZF-Screens BV. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Natural Killer (NK-) cells reveal a keen reaction to acute bouts of exercise, including changes of epigenetic modifications. So far, exercise-induced alterations in NK-cell DNA-methylation were shown for single genes only. Studies analyzing genome-wide DNA-methylation have used conglomerates like peripheral blood mononuclear cells (PBMCs) rather than specific subsets of immune cells. Therefore, the aim of this pilot-study was to generate first insights into the influence of a single bout of exercise on genome-wide DNA-methylation in isolated NK-cells to open the field for such analyses. Five healthy women performed an incremental step test and blood samples were taken before and after exercise. DNA was isolated from magnet bead sorted NK-cells and further analyzed for global DNA-methylation using the Infinium MethylationEPIC BeadChip. DNA-methylation was changed at 33 targets after acute exercise. These targets were annotated to 25 genes. Of the targets, 19 showed decreased and 14 increased methylation. The 25 genes with altered DNA-methylation have different roles in cell regulation and differ in their molecular functions. These data give new insights in the exercise induced regulation of NK-cells. By using isolated NK-cells, exercise induced differences in DNA-methylation could be shown. Whether or not these changes lead to functional adaptions needs to be elucidated. As shown by Bigley et al. [dim NK-cells, leading to changes in the distribution of NK-cell subsets. Data for NK-cell function is inconsistent but a tendency regarding higher NK-cell function after intensive exercise is reported [Natural killer (NK-) cells are a distinct cell population of the innate immune system that build the first line of defense against virus-infected and neoplastic cells . NK-cellreported .Research of the past decade suggests that several short- and long-term adaptions of various tissues in response to exercise are based on epigenetic modifications ,7,8,9, sKIR3DL1 gene was affected. In this trial, DNA-methylation was assessed using the high-resolution method targeted deep amplicon bisulfite sequencing (TDBS).Using isolated NK-cells, we  have shoSince immunohistochemically antibody-based assessment of DNA-methylation is relatively vague, and TDBS does not allow the analysis of a wide range of genes, this pilot-study aims to generate first insights into the influence of a single bout of exercise on gene specific DNA-methylation using a microarray approach in isolated NK-cells and open the field for such analyses.This study was performed in accordance with the declaration of Helsinki and was approved by the ethics committee of the University Hospital of Cologne (IRB#13-274). Five healthy women (age 61.4 ± 8.0 years) were recruited by regional newspaper announcement between December 2014 and September 2015 and provided written informed consent. The term “healthy” was defined as no acute or chronic diseases. Any kind of drug intake was an exclusion criterion. For analyzing the effects of a single bout of exercise on the methylome of NK-cells, venous blood samples were collected before (t0) and 1 min after (t1) an incremental step test on a bicycle ergometer.The incremental step test was performed on a bicycle ergometer  with a spirometry analysis . The incremental step test began with a 1 min rest measurement, followed by a 3 min warm-up at 50 watts power output and an increase of 25 watts every 2 min until exhaustion (Respiratory quotient >1). Heart rate  and self-perceived exhaustion  were assessed in each intensity step.Blood samples were collected before (t0) and 1 min after (t1) the incremental step test. Blood samples were used for isolation of peripheral blood mononuclear cells (PBMCs) with a lymphocyte separation medium  based density gradient centrifugation. Isolated PBMCs were used for a magnetic bead associated negative separation of NK-cells  according to the manufacturer’s protocol.DNA was isolated using a column-based isolation  according to the manufacturer’s protocol.® MethylationEPIC BeadChip  according to the manufacturer´s protocol. Methylation data was calculated with Genome Studio 2011.1 containing the methylation module . Methylation was displayed in β-values. The data discussed have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE129376 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE129376).Genome-wide DNA-methylation was assessed with the InfiniumFunctional annotation of genes showing changes in DNA-methylation was performed using the DAVID Bioinformatics Resources .bright and CD56dim NK-cells were gated and presented as percentage of the NK-cell pool.To control for changes of the NK-cell subsets distribution, isolated PBMCs were used for flow cytometry analysis. PBMCs were stained with anti-CD3 APC-C7 and anti-CD56 PE . Flow cytometry was performed on a BD FACS Array  and NK-cells were gated as CD3− and CD56+. Furthermore, CD56p-value correction according to Benjamini-Hochberg [p ≤ 0.05 as described by Tremblay et al. [p-value indicated whether the sample signal was distinguishable from the negative control. Targets indicating a detection p > 0.05 in more than 10% of the samples were excluded from analyses. Flow cytometry data was compared using a Wilcoxon signed-rank test. The level of significance was set at p ≤ 0.05.Genome Studio was used to perform a differential methylation analysis, to detect differences between before (t0) and after (t1) acute exercise. The illumina custom model, including consideration of the false discovery rate (FDR) by Hochberg , was usey et al. . In brie2peak) of 32.4 ± 3.8 mL/min/kg with a maximal power output of 165.0 ± 13.7 Watt. Detailed characteristics of the participants are shown in To analyze the influence of a single bout of acute exercise, five healthy women were recruited with an age of 61.4 ± 8.0 years. The incremental step test revealed a peak oxygen uptake (VOp ≤ 0.05. Comparing DNA-methylation before (t0) and after (t1) acute exercise revealed 33 targets being changed with a DiffScore ≥ |13| (0.004%). Within the 33 targets affected by acute exercise, 25 were annotated to a gene. As shown in Analysis with the Infinium MethylationEPIC BeadChip revealed 864,614 targets of a total of 865,918 (99.8%) that could be detected with a detection FASLG is the gene with the most functional annotation. It belongs to the plasma membrane and is involved in regulation of transcription.DAVID Bioinformatics Resources  was usedbright NK-cells within the NK-cell pool, decreased from 14.91% ± 4.64% to 10.38% ± 3.20% comparing before and after acute exercise. Accordingly, the percent of CD56dim NK-cell increased from 84.63% ± 4.78% to 89.12% ± 3.57%. Nevertheless, both changes did not reach statistical significance .Flow cytometry was used to control blood samples for changes in NK-cell subset distribution. The percent of CD56Investigating the effect of exercise interventions on DNA-methylation in specific immune cells is a novel research field and literature in this context is limited to studies analyzing PBMCs ,11,12,13KIR2DS4-gene. Comparing the KIR2DS4 and KIR3DL1 genes in both attempts, we found 6 CpGs (4 in the KIR2DS4 gene and 2 in the KIR3DL1 gene), which were analyzed in both attempts (TDBS and EPIC). All 6 commonly analyzed CpGs remained unaltered when comparing both methods and the ones that were changed in the previous study [Our results reveal a fast adaption of NK-cells to an acute bout of endurance exercise by changes of DNA-methylation at 33 targets. So far, DNA-methylation was thought to be a more stable epigenetic modification when compared to histone modifications or miRNAs. Nevertheless, our results indicate a more dynamic function of DNA-methylation in response to external stimuli, e.g., exercise, and this hypothesis is supported by recent research. It was shown that a single bout of exercise changes promoter DNA-methylation in muscle  and adipus study  were unfFASLG with the most annotations. FASLG codes for a ligand of the FAS-receptor and induces apoptosis upon binding, one mechanism of NK-cell cytotoxicity. Our results reveal a slightly reduced DNA-methylation of FASLG in one CpG within the first exon after acute exercise. As shown by Mooren et al. [Analysis of functional annotation reveals n et al. , gene exbright and CD56dim reveal different functional properties [dim NK-cells [The NK-cell subsets CD56operties  and may operties ). MoreovNK-cells  describedim and CD56bright) provided even more detailed information of differential regulation of the subgroups. However, the amount of DNA isolated from NK-cell subpopulations would be a bottle neck for those analyses. Moreover, the timing of blood sampling after exercise affected the composition of circulating leukocytes and was standardized precisely. By using venipuncture, each blood sampling could be subject to a minor delay and therefore may have influenced the results. Since a change in NK-cell subset proportions was absent, exercise bouts of longer durations should be used to increase the influence of exercise on the NK-cell pool. Furthermore, more measurement time points could give information about the kinetics of exercise induced changes of DNA-methylation and should be considered in following studies. Gene expression of affected genes is not measured. Consecutive studies are warranted, analyzing whether changes in DNA-methylation correspond to changes in gene expression of the affected genes.The results of the presented study should be considered within the context of its limitations. Chip based microarrays for DNA-methylation analyses are a state-of-the-art method and allow the quantification of DNA-methylation at a vast number of single CpGs in parallel. Especially, the Illumina MethylationEPIC BeadChip is described as a robust and reliable platform . NeverthIn conclusion, this is the first study showing effects of a single bout of endurance exercise on genome-wide DNA-methylation in isolated NK-cells. Acute exercise affects the functional genome of NK-cells by changes in DNA-methylation. In order to investigate such changes, it is proposed to use specific cell types rather than conglomerates like PBMCs. More research is needed to combine changes in the functional genome of NK-cells with changes in NK-cell function.


Anxiety disorders are highly prevalent in adolescence, but access to health care services is limited and only few receive professional help. Internet-based cognitive behavioral therapy (ICBT) has been proposed to increase accessibility and reduce costs of treatment.The study evaluated the efficacy of a Danish version of the guided ICBT program ChilledOut Online, developed at the Centre for Emotional Health, Macquarie University, Australia.At the Centre for Psychological Treatment of Children and Adolescents, Aarhus University, Denmark, a randomized controlled trial was conducted with 70 adolescents (13–17 years) with anxiety disorders according to DSM-IV. Participants were randomly assigned to a 14-weeks therapist-guided ICBT or to a waitlist condition. Outcomes were evaluated post-treatment and at 3- and 12-month follow-up.At post-treatment, the ICBT group significantly outperformed the waitlist condition with moderate to large between-group effect sizes on diagnostic severity and anxiety symptoms rated by clinicians, and by adolescents and their parents. Forty percent of adolescents in ICBT were free of their primary diagnosis compared to 16% in the waitlist condition. Treatment gains were maintained at 3- and 12-month follow-up.Results of the study provide support for the efficacy of guided ICBT for adolescents with anxiety disorders.NCT02535403.ClinicalTrials.gov:  Anxiety disorders are common in adolescence with 5–12% of youths from western countries suffering from the disorder . When leInternet-based CBT (ICBT) has been proposed as a means to increase access to, and reduce costs of, psychological interventions. ICBT typically provides therapeutic content comparable to that of regular face-to-face CBT and is typically presented in modules on a weekly basis , 21. ICBICBT for adults has been widely researched with substantial evidence of effectiveness for a range of anxiety disorders , 26–30. g = 0.53–1.41). None of the above mentioned meta-analyses examined the effect of ICBT for adolescents specifically, but those that investigated age as a moderator of outcome  Child ve et al. . Lenharde et al.  with an the ChilledOut Online program [Chilled [the Cool Teens program [The present study investigates a Danish version of  program  develope[Chilled  for adol program , 55, has program . An init program .The present study examined the efficacy of ChilledOut Online using a randomized waitlist-controlled design with follow-up assessments at 3- and 12-month post-intervention. We hypothesized that the ICBT intervention would demonstrate superiority to the WL condition primarily by reducing diagnostic severity and anxiety symptoms, but also by reducing symptoms of depression and improving self-efficacy and quality of life. Additionally, we expected these outcomes to be maintained at follow-ups.The study took place at the Centre for Psychological Treatment of Children and Adolescents (CEBU), a research and teaching facility at the Department of Psychology and Behavioral Sciences, Aarhus University, Denmark. The study was approved on May 7, 2015 by the local Ethics Committee of Central Denmark Region (1-10-72-98-15) and by the Danish Data Protection Agency. Furthermore, the trial was registered within ClinicalTrials.gov, registration number: NCT02535403.Diagnostic and Statistical Manual of Mental Disorders  13 to 17 years of age; (b) a primary anxiety disorder diagnosis according to the  DSM–IV; ); (c) di DSM–IV; ); (b) suParticipants were recruited from May 15 to December 18, 2015 and the last follow-up assessment was obtained by March 1, 2017. Although the recruitment process was initiated prior to trial registration , baseline assessments and enrolment was not commenced until registration had been confirmed. Families had referred themselves to the CEBU secretary in response to postings on the website or recommendations from local community health services. Interested families were invited to send in a brief description of the adolescent’s major problems. If deemed eligible for the present study, families were contacted for a semi-structured diagnostic telephone interview and sent a package of online questionnaires (see full description under Measures). Those who met inclusion criteria were given verbal and written information about the study procedures. After adolescents and their parents provided written consent, they were included in the study and randomized to condition. The randomization sequence was created with an online computer random number generator using permuted block design with a fixed block size of 10 at a 1:1 allocation ratio to the ICBT or the WL condition. The sequence list was kept concealed from researchers and therapists, stored by an external secretary at the University who administered group assignment to included participants according to the randomization sequence. Participants in both conditions were encouraged not to engage in other forms of treatment nor make changes to their use of psychiatric medication during the acute treatment and waitlist period.Adolescents randomized to the WL condition were instructed to wait 14 weeks for treatment. During this period, participants did not have any form of planned contact with the project team. After 14 weeks, adolescents and parents recompleted the assessment measures, took part in a second telephone interview, and were offered the ICBT treatment. Adolescents randomized to the ICBT condition were informed of their allocation over the phone by their appointed therapist and provided introductory information about the program. During this 20-minute introductory phone call, the course of the intervention was summarized, adolescents were given a brief introduction to the program website and functions, and therapists and adolescents agreed upon the time and dates of their weekly supportive phone calls. The adolescent then received a letter outlining the treatment start date and program information, i.e. the program URL, a personal username, a temporary password, the therapist e-mail address, a photo of the therapist, and a calendar plan of the future therapist phone calls. All therapist calls were recorded using Crystal Gears® Ver. 2.00 RTM for supervision purposes and to ensure integrity of the intervention.Adolescents’ diagnostic status was assessed at baseline (pre), after the intervention (post), and at three-month follow-up with the ADIS-IV C/P  administClinician-rated diagnostic severity. The ADIS-IV C/P [κ = 0.80 . The intraclass correlation coefficient , was fair for the CSR of the primary anxiety diagnosis (CSRprim), ICC = 0.419  and good for the CSR of all anxiety diagnoses , ICC = 0.731 .S-IV C/P  is a semS-IV C/P . In addiS-IV C/P . ConcurrS-IV C/P . AdminisS-IV C/P . The intAdolescent- and parent-rated anxiety symptoms. The Spence Children’s Anxiety Scale  for the total score was good to excellent for adolescent (α = 0.88), mother (α = 0.89), and father (α = 0.90) report versions of the scale.e  measuree  was acceptable for adolescent reports (α = 0.77), and good for mother and father reports .  measure  was excellent for adolescent report (α = 0.90), and good for mother and father reports .  measure  for the total scale was good, α = 0.89.  is a 24  in the present sample was good, α = 0.86.  is a fi .Treatment satisfaction. Satisfaction with the ICBT intervention was measured in adolescents and parents post treatment using a short questionnaire adapted from The Experience of Service Questionnaire [ionnaire , 73. SepProgram activity and support. Adolescents’ program activity was automatically registered at the website server as number of logins, number of module visits and number of module components activated. Therapists kept a record of all contact with adolescents and parents, noting the duration of each phone call and all e-mail correspondence throughout the intervention. Moreover, adolescents were asked to rate their average time spent per week on program relevant activities (on- and offline separately) at post treatment, and parents were asked to report their average time spent per week helping their child with program relevant activities (on- and off-line combined).The Strengths and Difficulties Questionnaire [The Working Alliance Inventory-Short Form [The following measures were administered but not reported here: ionnaire , 75; Theort Form .ChilledOut Online is a treatment program based on the Cool Kids and Chilled anxiety management program developed at Macquarie University, Sydney, Australia . The proAdolescents received a weekly phone call from their therapist set to a duration of approximately 20 minutes, with a focus on assisting learning of program skills and strategies, offering technical assistance, giving feedback about homework tasks, and encouraging program use during times of low motivation. The therapists were graduate psychology students in a clinical training course supervised by psychologists working at CEBU. All therapists received training in cognitive behavioral therapy and a thorough introduction to the program. Moreover, all therapists were equipped with a semi-structured “manual” with topics to be dealt with in the calls. The therapists had access to participants’ module activities and worksheets throughout the program. At three-month follow-up, adolescents received a booster phone call from their therapist with the aim of consolidating previously learned skills.the ChilledOut Parent Companion describing their role in treatment and providing them with an explanation of the program’s core treatment strategies . Furthermore, the handout included advice on how to support their teenager in his/her efforts to overcome anxiety. Parents are encouraged to lend support and motivational encouragement to their child during treatment. However, they are also advised to be sensitive to their child’s need for autonomy. Within the first two weeks of the intervention, parents received a brief phone call in which the therapist introduced him- or her-self and answered any questions about the program. During the remaining intervention period, parents were invited to contact the therapist through e-mail and, if needed, they were offered a short phone call from the therapist. Based on parent feedback from a previous feasibility trial of the ChilledOut Online program [Shortly before treatment start, parents received  program , parentsd = 0.80) with a power of .80 and two-tailed α = .05 [Allowing for approximately 20% dropout (cf. ), the st α = .05 .t-tests, χ2-tests or Fisher’s exact test (two-sided) as appropriate. Pre–post comparisons between the two conditions were analyzed as (1) number of participants free of primary or all anxiety diagnoses at post-treatment; (2) degree of change on continuous outcome measures from pre to post; and (3) number of participants with clinically significant change on the SCAS.Baseline group differences were explored using independent samples p = 0.017 (0.05/3) for each set of analyses.Mixed linear models (MLMs) were used to compare the two conditions from pre to post on continuous outcome variables. MLMs tolerate missing values and thus do not unnecessarily compromise statistical power. All MLMs were based on the intention-to-treat sample (N = 70) without any ad hoc imputations of missing values, which is recommended over other procedures of handling missing data in longitudinal clinical trials . All MLMrxx is the reliability of the measure . The RCI was calculated using standard deviations and internal consistency ratings from Danish norm data [cut-off) defined as the mid-point between the clinical and non-clinical population, was calculated with the formula: CScut−off = ((Mclin×SDnorm)+(Mnorm×SDclin))÷(SDnorm+SDclin). The CScut-off was calculated from Danish community and clinical norms split into gender and age groups reported in Arendt et al. [Using Jacobson and Truax’s  method, orm data . The clit et al. . As suggt et al. , particiOnly participants randomized to the ICBT condition were included in the follow-up analyses. Analyses of treatment outcome maintenance were conducted on SCAS, CALIS and WHO-5 from pre to 3- and 12-month follow-up and for ADIS CSR from pre to 3-month follow-up using MLMs. For models including more than two measuring points , the slope was specified as random if it significantly improved model fit as evaluated by a change in the –2LL fit statistics .d, with 0.2, 0.5, and 0.8 considered a small, medium, and large ES, respectively [d was derived by the following formula: d = (MdiffICBT÷SDICBTpooled)−(MdiffWL÷SDWLpooled), were Mdiff ICBT and Mdiff WL are the means of the pre-post difference scores for the ICBT and WL group respectively, and SDICBT pooled and SDWL pooled are the pooled standard deviations of the two groups, calculated by the formula: d = (Mpre−Mpost)÷SDpooled.For all analyses ES calculations were based on observed values and expressed as Cohen’s ectively . For theStudy dropouts were defined as participants who had not remained in contact with the therapist for the entire 14 weeks of intervention. Otherwise participants were considered completers, regardless of their number of completed modules or amount of completed therapist calls.All statistical analyses were carried out using IBM® SPSS® statistics, v.24.0 .A total of 120 adolescents were recruited. Eighty-four adolescents presented themselves with eligible problems and thus received questionnaires and phone interviews  dropped out prior to post-assessments. Two participants (ICBT) dropped out due to loss of motivation, one (WL) experienced a worsening in symptoms during the waitlist period , and two (WL) gave no explanation. No significant differences were found on primary outcome pre-treatment scores (CSR and SCAS-C/P), gender, age, number of anxiety diagnoses, age at anxiety onset, previous therapeutic treatment, primary diagnosis or computer experience between dropouts and completers. Ninety-six percent (63/65) of the adolescent study completers returned the self-report questionnaire at post, while 100% (65/65) took part in the diagnostic post-interview. At 3-month follow-up, 82% (27/33) of the adolescent study completers (ICBT condition only) returned the self-report questionnaire, while 85% (28/33) completed the diagnostic interview. At 12-month follow-up, 76% (25/33) of the adolescent study completers returned the self-report questionnaire. Reasons for non-completion of questionnaires and interviews are largely unknown, as most of these participants could not be reached.t (68) = 3.345, p = 0.001., where families in the WL condition had higher income than families in the ICBT condition.Baseline characteristics are presented in Regarding computer experience, thirty-three (94%) of the adolescents randomized to the ICBT condition reported feeling either ‘fairly confident’ or ‘very confident’ using computer and internet. Two (6%) reported feeling only ‘a little confident’ and none reported ‘not at all confident’.Participants  completed a mean of 5.4  of the eight modules available. Modules were registered as completed if participants had activated ≥ 80% of core module components . According to website server logs, participants activated a mean of 74%  of all module components. Total number of logins to the program web site ranged from seven to 51 . χ2(1) = 4,89, p = .027 (χ2(1) = 7,89, p = .005. The odds of a participant being free of all anxiety diagnoses were 12.38 times higher if they received ICBT than if they were on WL.At post-treatment, of those with diagnostic post-interview data significantly more adolescents in the ICBT condition (40%) were free of their primary diagnosis, compared with adolescents in the WL condition (16%), p-values for all outcome scales at pre and post for the ICBT and WL condition and at follow-up for the ICBT condition. The number of adolescents in the ICBT group free of their primary diagnosis at post-treatment (intention-to-treat sample) was distributed as follows: SAD = 0% (0/2), SoP = 21% (3/14), SP = 50% (2/4), PD = 67% (2/3), PD with AP = 0% (0/1), AP without PD = 0% (0/2), GAD = 83% (5/6), OCD = 67% (2/3). Although numerically diverse, a Fisher’s Exact test with eight groups  revealed a non-significant association (p = .062) between type of primary anxiety diagnosis pre-treatment and whether or not the adolescents were free of these post-treatment .prim; d = 0.65, p = 0.022) and all diagnoses  , mothers  and fathers , indicating significantly greater reductions in adolescent anxiety symptoms in the ICBT group compared to WL.Pre-post MLM analyses revealed significant effects on clinician-rated severity of primary diagnosis , mother reports on symptoms of depression  and adolescent report on the emotional subscale of self-efficacy . For the remaining secondary outcome measures , no significant interaction effects were found  = 11.62, p = .001). Also, significantly more participants in the ICBT condition (44%) than in the WL condition (6%) were classified as recovered (χ2 (1) = 11.56, p = .001) according to adolescent SCAS-C report. For the post-treatment mother-reported SCAS-P, significantly more participants in the ICBT condition (69%) than in the WL condition (22%) were considered improved (χ2 (1) = 14.66, p < .001). There were, however, no significant differences in number of participants classified as recovered (χ2 (1) = 4.62, p = .032) in the ICBT condition (26%) compared to the WL condition (6%). For father reported SCAS-P, the number of participants improved post-treatment in the ICBT condition (35%) was not significantly larger than those improved in the WL condition  = 2.02, p = .156). Similarly, participants classified as recovered according to father reported SCAS-P at post-treatment did not differ significantly (Fisher’s exact test: p = 1.00 (two sided)) in the ICBT group (4%) compared to the WL group (7%).According to adolescent reported anxiety symptoms on SCAS-C, significantly more participants in the ICBT condition (69%) than in the WL condition (26%) were classified as improved at post-treatment , while analyses from post to 3-month follow-up revealed small and non-significant changes .At 3-month follow-up, 36% adolescents were free of their primary anxiety diagnosis and 30% were free of all anxiety diagnoses. MLM within-group analyses of the CSRd = .99, p < .001; mothers: d = 1.10, p < .001; and fathers: d = .94, p < .001). Analyses from post to 3-month follow-up revealed significant improvements for the father report on SCAS-P . Otherwise, analyses generally showed non-significant positive changes in the follow-up periods.Within-group analyses of the SCAS reports from pre to 12-month follow-up see  revealedAt 3-month follow-up, the number of adolescents classified as improved on the SCAS was 67% according to adolescent report, 70% according to mother report and 61% according to father report. The number classified as recovered was 52% according to adolescent report, 39% according to mother report, and 30% according to father report. At 12 month follow-up, the number of adolescents categorized as improved on the SCAS was 48% according to adolescent report, 65% according to mother report, and 67% according to father report. The number classified as recovered was 40% according to adolescent report, 45% according to mother report, and 43% according to father report.d = 0.50, p = 0.002; mothers: d = 0.868, p < 0.001; and fathers: d = 0.931, p < 0.001). Analyses of participants’ emotional well-being scores on the WHO-5 indicated no change, as the total treatment effect from baseline to 12-month follow-up was small and non-significant . Similarly, effects for both follow-up periods analyzed separately were small and non-significant  out of a maximum score of 21. Seventeen (55%) rated the statement “the treatment helped me” to be ‘true’, nine (29%) to be ‘partly true’, and five (16%) not to be true. Eighteen adolescents (58%) would recommend the treatment to a friend, four (13%) would not recommend it, and the remaining nine (29%) answered ‘partly true’ to the statement. Adolescents’ qualitative comments on the treatment were generally highly positive. Two (7%) would, however, have liked more time, and one (3%) expressed the need for face-to-face sessions with the therapist. Mothers and fathers were also generally satisfied with the intervention with a mean rating of 23.1 (SD = 3.66) and 22.2 (SD = 3.19), respectively, out of max 27.Participants and their parents were also asked post-treatment whether the treatment had caused them/their child to feel worse. One adolescent (3%) rated the statement to be true, while three adolescents (10%) rated it to be ‘partly true’. One mother (3%) rated the statement to be true, while two mothers (6%) answered ‘partly true’ to the statement. No fathers rated the statement to be ‘true’, but three (12%) rated it to be ‘partly true’. Regarding those (n = 2) who rated the above statement to be true, additional qualitative information from questionnaires and booster-sessions was collected but did not indicate requirements of further clinical intervention.The present study evaluated the efficacy of therapist-guided ICBT for adolescents with anxiety disorders. To the best of our knowledge, the study is the first randomized evaluation of the ChilledOut Online program, and it is the first systematic evaluation of an ICBT intervention for this population in Denmark.prim and CSRall in the present study  are in the lower end of those found in other similar WL controlled studies; for instance by Lenhard et al. [d = 0.69; and Vigerland et al. [d = 1.66. In their study of the Cool Teens CD-ROM, Wuthrich et al. [d = 1.35. The within-treatment pre-post effects on diagnostic severity were, however, quite similar in our study to that of other studies; for instance a d = 1.48 on CSRprim compared to ds 1.32–1.69 [d = 0.83. Seven of the 35 WL participants (20%) reported having had contact with mental health services during the WL period, three had received hypnosis in private practice, one CBT with the GP, and three had contacted the local psychiatric hospital, possibly explaining part of the improvements found in the WL group. Large changes in waitlist participants is however not unprecedented as previous studies of CBT with youth/children has found similar outcomes . The between-group ES’s found for CSRd et al. : d = 0.6h et al.  showed a.32–1.69 , 83, 84.s . Despitd = 0.68; SCAS-Pmotherd = 1.12; and SCAS-Pfatherd = 0.46. Moderate between-group treatment effects for child and adolescent self-rated anxiety symptoms on SCAS-C has previously been shown [d = 1.12) is, on the other hand, larger than those reported in previous studies . It16–0.69;  with motd = 1.39). As was found in this study, it is not unusual to find smaller outcomes (and/or less frequently significant findings) on depression than on anxiety symptoms , mother ratings of anxiety life interference (CALIS) and self-ratings of the emotional self-efficacy sub-scale . Two previous ICBT studies treating adolescents with anxiety disorders also measured depressive symptoms, with one  showing s  in studSupporting our third hypothesis, results at 3- and 12-month follow-up assessment demonstrated a maintenance of treatment effects for all included measures. These results are similar to those of Wuthrich et al. , Spence The study has a number of limitations that should be considered when interpreting the results. Although improvements in the ICBT group suggests that recovery could be attributed to the specific intervention and not to confounding factors such as spontaneous remission or repeated assessment, an active control condition would be required to rule out potential effects of such unspecific factors. The study was not controlled in the follow-up period since participants in the WL condition received treatment during this period. Rater blindness to treatment condition was often broken post-treatment and at follow-up. Moreover, the use of self-referral and the high socioeconomic status of participant families could have implications for generalizability of results, although it is difficult to predict the direction of potential effects. Lastly, the majority of CSR ratings were based on ADIS interviews conducted by students. Although thoroughly trained and supervised, they had only modest prior assessment experience compared to trained psychologists used in other studies. Combined with low variability in CSR of primary diagnosis  and the use of telephone-recorded interviews in the reliability checks, this might help explain the moderate inter-rater reliability scores found in the study.This study demonstrated the Danish version of ChilledOut Online to be efficacious and feasible in relieving symptoms of anxiety in adolescents. As such, the study supports previous findings of similar guided ICBT interventions and helps build a strong foundation for future research in and implementation of ICBT in mental health services for adolescents with anxiety disorders.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.


Their distinctive characteristic is the covalent attachment of heme to their polypeptide chain. This post-translational modification is performed by a dedicated protein system, which in many Gram-negative bacteria and plant mitochondria is a nine-protein apparatus (CcmA–I) called System I. Despite decades of study, mechanistic understanding of the protein–protein interactions in this highly complex maturation machinery is still lacking. Here, we focused on the interaction of CcmC, the protein that sources the heme cofactor, with CcmE, the pivotal component of System I responsible for the transfer of the heme to the apocytochrome. Using in silico analyses, we identified a putative interaction site between these two proteins , and we validated our findings by in vivo experiments in Escherichia coli. Moreover, employing NMR spectroscopy, we examined whether a heme-binding site on CcmE contributes to this interaction and found that CcmC and CcmE associate via protein–protein rather than protein–heme contacts. The combination of in vivo site-directed mutagenesis studies and high-resolution structural techniques enabled us to determine at the residue level the mechanism for the formation of one of the key protein complexes for cytochrome c maturation by System I.Cytochromes  How49 and CcmE Arg104 by using site-directed mutagenesis on the entire E. coli System I operon and examining the levels of holo-CcmE and holocytochrome c matured in vivo. We initially generated single-amino acid substitutions, where each of the two amino acids was replaced by an alanine. Surprisingly these mutations did not seem to have a very strong effect on System I function. Whereas holo-CcmE levels were slightly elevated for the Q49A-CcmC variant, they remained unaffected for the R104A-CcmE variant. In addition, holocytochrome c (holo-c550) levels were comparable with the WT System I for both variants . Taken together, these results suggest that these residues are not essential for the formation of a functional CcmC–CcmE complex.We sought to assess the role of CcmC GlnE. coli CcmC and CcmE and identified conserved amino acids that could mediate protein–protein interactions and were in the same part of the proteins as the residues identified by the covariance analysis. For E. coli CcmC, Asp47 , Gln50 (∼65% conservation), and Arg55 (it is either an Arg or a Lys) were plausible candidates . For E. coli CcmE, Asp101  and Glu105  were chosen . The rationale behind considering these amino acids was their high conservation and their ability to form electrostatic or hydrogen bond interactions. As only two putative interacting amino acids could be selected for CcmE, we also examined the solution NMR structure of this protein  as a potential candidate. Although this residue is not near Arg104 in the primary sequence , it is located near it and near Asp101 and Glu105 in the 3D structure of apo-CcmE (see B).Reasoning that the amino acids involved in the interaction surface of the two proteins could be near the covarying pair, we turned to bioinformatics. We searched in bacterial and archaeal representative proteomes for homologs of  protein  and searCcmE see B.E. coli CcmC and CcmE was tested in vivo using variant System I operons where each of the six identified residues  was replaced by an alanine. We observed identical results for the maturation of holo-CcmE and holo-c550 for all tested variant operons. Holo-CcmE decreased significantly (lanes 2–4 in A and B), top gel strip) compared with the level matured by the WT System I (seen in lane 1). Holo-c550 amounts were also significantly reduced (lanes 2–4 in A and B), middle gel strip), reflecting the decrease in holo-CcmE. Total CcmC  and CcmE  levels in all variants were assessed by immunoblotting and were found to be unaffected by the amino acid replacements.The presence of an interaction site between A and B), top). We carried this out by mutating residues in CcmE  and found that production of holo-CcmE and holo-c550 was entirely abolished , whereas the total CcmE amount remained unaffected . Thus, we can conclude that the interactions between Asp47, Gln50, and Arg55 of CcmC and Arg73, Asp101, and Glu105 of CcmE are collectively essential for the successful association of the two proteins, leading to maturation of CcmE and cytochrome c.The results from the single-amino acid substitutions indicate involvement of these residues in the interaction of CcmC with CcmE. To assess their collective role, we investigated whether removal of all three of them on one of the protein partners would abolish the residual holo-CcmE amount obtained with the single variants (49 in CcmC and Arg104 in CcmE)  unaffected compared with the amounts generated by the WT System I (seen in lane 1). However, a double-alanine mutant led to a dramatic outcome; substantial accumulation of holo-CcmE was observed , but holo-c550 production was entirely abrogated . Total CcmE amounts were assessed by immunoblotting for all constructs and were found to be unaffected .After validating the CcmC–CcmE interaction site, we turned back to the residues identified by the covariance analysis (Glnin CcmE) , whose r49 and CcmE Arg104 are likely playing an important role in the release of holo-CcmE from the CcmC–heme–CcmE complex. To further explore this hypothesis, we generated a series of System I variants and examined the amount of holo-c550 produced in vivo; in this experimental setting, WT levels of holo-c550 indicate that the substitution in question does not affect the CcmC–CcmE interaction and System I function. The mutations that were assessed are summarized in Fig. 3B). Several other mutations , also did not affect System I function. We were only able to reproduce the result observed with the double-alanine substitution (A) when we replaced one or both alanines in this variant with a valine residue . For these mutants, we observed holo-CcmE accumulation and lack of holo-c550 production (top and middle gel strips), whereas total CcmE amounts remained unaffected (bottom gel strip).The effect observed for the double-substitution is strongly reminiscent of the phenotype of the K40D-CcmA mutation, abolishing the ATPase activity of CcmA , 27. Thiutations , includititution A when we49 and CcmE Arg104 is not clear. These residues are likely to interact, confirming the prediction by Ovchinnikov et al. (B). Surprisingly, double serine or cysteine substitutions (C), which are similar in side-chain volume to the double alanine or alanine/valine substitutions, did not elicit the phenotype observed in Fig. 3A (lane 4) and Fig. 3D (lanes 2–4) either, suggesting that side-chain size alone is not the determinant of the interaction. In the absence of a structure for CcmC, it is difficult to propose an exact function for the Gln49-CcmC-Arg104-CcmE pair. Nonetheless, we can suggest that these residues could play a steric role that would ensure that other residues in the CcmC–CcmE interaction surface are optimally placed for the release of holo-CcmE after heme attachment.Considering the outcome of our mutagenesis experiments , the exav et al. . Howeverv et al. B. Surpriitutions C, which E. coli CcmC and CcmE from bacteria and archaea (Table S2) shows that they can be occupied by a broad range of residues; however, the combinations that we identified as ones that are detrimental to System I function were not found to occur. We tested one of the alternative combinations that we identified bioinformatically for this pair (Q49K-CcmC/R104A-CcmE-System I variant) and found it to behave like the WT System I in terms of holo-c550 production , the CcmE interaction site does not coincide with the heme-binding site of CcmC, although it is sufficiently close for a productive subsequent interaction. The same can be concluded when the CcmE interacting residues are mapped on the 3D structure of the protein (B); these amino acids along with the covarying Arg104 form a tight cluster located on a flexible loop placed above His130, the E. coli CcmE heme-binding residue.Our sumption . The strsumption . Based otion , were postulated to form a heme-binding site. Some of these findings were validated in vivo in another study, using a minimal experimental system expressing only CcmC and CcmE  E. coli CcmE bearing a C-terminal His6 tag). However, instead of purifying the holoprotein after co-expression of soluble CcmE with System I, we purified it in its apo-form and fully reconstituted it with heme in vitro. The advantage of this approach is that we were certain that our sample contained solely WT holo-CcmE; we generally find that separation of mixtures of holo- and apo-CcmE produced in vivo is particularly challenging and never occurs fully. After staining our protein for covalently bound heme, we further characterized our sample by visible absorption spectra with and without hydroxide and pyridine in reducing heme conditions and found it to have the same heme attachment as in naturally occurring CcmE   spectra of 15N-labeled apo-CcmE and 15N-labeled oxidized holo-CcmE  and assessed the effect of heme binding by comparing individual peak intensities for all backbone residues. Proximity of an amino acid with the paramagnetic heme should lead to peak broadening and decrease in the peak height ; therefore, a lower peak intensity ratio should be observed for residues affected by the heme (A). We found that residues in the core of the protein showed a uniform level of nonspecific broadening that is to be expected because of the presence of the heme in the sample. Pronounced broadening was only observed at the C terminus, especially after residue 139. Whereas peaks for some of the residues cannot be observed due to very low intensity  or cannot be included in the graph because of peak overlap , it is clear that the residues postulated by Enggist et al.  . In addi  was due to the presence of the affinity tag, we also produced an in vitro heme-reconstituted CcmE sample devoid of the His6 tag. This was challenging because the His6 tag, although not essential, is useful for efficient heme attachment to the protein . This indicates that when heme is covalently bound to His130 in CcmE, it has very few specific interactions with the protein backbone.The sample we examined , had a  protein A was due protein , but it oadening B. This i6-tagged samples by {1H}-15N heteronuclear NOE experiments . For both samples, residues in the β-sheet structure generally gave values higher than 0.75, indicating a rigid backbone. Residues with NOE values lower than 0.75 are either located at the N terminus, which is expected as this protein is linked to a transmembrane helix in the cell, or at the C terminus. At the C terminus, residue 128 is the first amino acid with a reduced NOE value (0.68), and although we cannot measure values for residues 129 and 130, Asp131 has a significantly lower NOE ratio of 0.49, suggesting that the protein is likely flexible from residue 128 onward. The fact that the results were similar for both apo- and holo-samples also suggests that the presence of the attached heme does not alter the flexibility of the protein.Finally, we tested the overall flexibility of the protein backbone with and without attached heme using our initial Hism), the addition of heme led to rapid (<10-min) covalent heme attachment to the protein . This occurred even when we used WT apo-CcmE devoid of its C-terminal His6 tag , which has been shown previously to be slower in covalently attaching heme in vitro , and once more we found that there is no specific broadening in the structured core of the protein that would indicate a heme-binding site (C). We note that comparison of these results with the graph obtained for cleaved WT holo-CcmE (B), shows that there is more specific broadening at the C terminus of H130A-CcmE. Nonetheless, most of the broadening is around Arg149, and very little broadening is observed for residues 130–133, which are the site of covalent heme attachment in the WT protein.We found that there is no heme-binding site on the structured core of holo-CcmE. However, this species occurs after covalent heme attachment and could well not require any further interaction with the heme moiety. The most obvious way to assess the noncovalent interaction of apo-CcmE with heme would be to perform NMR experiments on a mixture of WT apo-CcmE and heme, soon after the heme addition. Unfortunately, we found that for protein concentrations required for NMR spectroscopy  . We found that replacement of each of these amino acids with alanine led to significant decrease in holo-CcmE and holocytochrome c formation (A and B), whereas the absence of all three residues on one of the interacting partners abolished System I function entirely (C). The latter proves that each of the identified residues contributes to, and that they are collectively essential for, the interaction of CcmC with CcmE. The fact that production of holocytochrome c mirrored holo-CcmE maturation for each of the variants (A and B), further demonstrates that these residues affect the association rather than the dissociation of these two proteins. In addition, because single-amino acid substitutions on either side of the interacting surface (CcmC or CcmE) have the same effect (A and B), these six residues are potentially organized into three interacting pairs. Based on the nature of the identified amino acids, one of these interacting pairs likely involves Asp47 in CcmC and Arg73 in CcmE.The purification of the complex between heme-bound CcmC and CcmE , 24, prov et al.  pinpointormation , A and Bentirely C. The lavariants , A and Be effect , A and B73, Asp101, and Glu105) on the structure of CcmE (78 and Ile98 (magenta), are sufficiently surface-exposed and have side chains with the right orientation to be part of it. These are nonpolar residues. Therefore, if indeed they were involved in the interaction of CcmE with CcmC, they would most likely contribute weaker van der Waals interactions with their partners on CcmC. In the absence of a structure for the latter, it is not possible to venture a guess regarding which CcmC residues Leu78 and Ile98 of CcmE could be interacting with. We also note here that the six residues we identified are likely not the sole determinant of the overall CcmC–CcmE interaction. Whereas they are certainly extremely important for the association of the periplasmic domain of apo-CcmE with the periplasmic portion of heme-bound CcmC, as confirmed by the dramatic phenotypes observed by their substitution . Replacement of these residues with alanines or valines led to impressive accumulation of holo-CcmE and the complete absence of holocytochrome c formation, strongly reminiscent of the K40D-CcmA-System I phenotype , which show that although there is a significant variation in the amino acids found at these positions in related bacterial species, the combinations leading to the phenotype we observed  are never found. The proposed steric role for these residues makes sense if one considers that although CcmC and CcmE interact with high affinity . CcmE associates with heme-bound CcmC via three pairs of interacting residues  forming a stable CcmC–heme–CcmE complex. During this association, Gln49 and Arg104 in CcmC and CcmE, respectively, also interact, exerting a steric role within the CcmC–CcmE interaction interface that prevents the two proteins from binding too tightly to each other and makes the subsequent release of holo-CcmE possible (step 2). In the CcmC–heme–CcmE complex environment, the CcmC–CcmE interaction places CcmE in an optimal position for covalent heme attachment. Its flexible tail is brought into proximity of the heme moiety by the interaction interface and is likely further oriented toward the heme by Tyr134 (step 3). Tyr134 is almost entirely conserved , and, although not essential, it has been shown to be important for efficient holo-CcmE production . Despite important recent progress in this research direction  using plasmid pE221 . The resulting expression vector encodes an N-terminal polyhistidine tag, followed by the immunity 9 gene and a Factor Xa cleavage site before the multiple cloning site of TAGZyme pQE. pCcmC1 was generated by amplifying E. coli ccmC from pEC86 using oligonucleotides P3 and P4 and ligating it into NdeI-HindIII–digested pQE2-Im9 (Table S3). DNA manipulations were conducted using standard methods. Oligonucleotides were purchased from Sigma Genosys. KOD Hot Start DNA polymerase (Novagen) was used for all PCRs, enzymes were purchased from New England Biolabs, and all constructs were sequenced before use.Plasmids and oligonucleotides used in this study are listed in id pE221  or pEC86id pE221  as templTable S1. Cell growth and fractionation for in vivo experiments were carried out as follows. Experiments were performed in the WT E. coli strain JCB38725 . Paracoccus denitrificans cytochrome c550 was co-expressed with the E. coli ccm operon (or its variants) from plasmid pKPD1 , specifically for assessing total CcmC amounts. In all cases, cell growth was conducted in 200 ml of 2×TY medium  in 2.5-liter flasks; the small culture volumes ensured sufficient aeration, preventing expression of the endogenous E. coli ccm genes. Cultures were inoculated from single colonies and incubated at 37 °C for 15–18 h with shaking at 200 rpm. 1 mm isopropyl-1-thio-β-d-galactopyranoside was added to the cultures from inoculation. 100 μg/ml ampicillin and 34 μg/ml chloramphenicol were used when appropriate. For the extraction of periplasmic fractions, cells were harvested and spheroplasted as described by Allen et al. (g for 1 h at 4 °C. The membrane fraction was resuspended in ∼25 ml of 50 mm Tris-HCl (pH 7.5), 150 mm NaCl and was centrifuged again as above. The washed crude membrane fraction was resuspended in 1–2 ml of 50 mm Tris-HCl (pH 7.5), 150 mm NaCl.Bacterial strains used in this study are listed in JCB38725 . The E. n et al. . For theE. coli BL21 (DE3) (Stratagene) were transformed with plasmid pE221 (Table S3) expressing leaderless (Ser32–Ser163) E. coli CcmE. Transformants were initially grown on lysogeny broth  agar plates supplemented with 100 μg/ml ampicillin. For production of unlabeled protein samples, transformants were subsequently used to inoculate small-scale overnight cultures (5 ml of LB) in 50-ml polypropylene tubes, supplemented with 100 μg/ml ampicillin, and incubated at 37 °C with shaking at 200 rpm for 15–18 h. 500 ml of LB in 2.5-liter flasks were then inoculated from the overnight cultures (1:250), and the resulting suspensions were incubated at 37 °C with shaking at 200 rpm until A600 = 0.8. 1 mm isopropyl-1-thio-β-d-galactopyranoside was used for protein induction, and cultures were further grown at 30 °C with shaking at 200 rpm for 15 h. For the production of 15N-labeled protein samples, cell growth and protein induction were carried out as described in previous work  charged with Ni2+ and equilibrated with 50 mm Tris-HCl (pH 7.5), 300 mm NaCl. The column was washed with the equilibration buffer, and apo-CcmE was eluted with 50 mm Tris-HCl (pH 7.5), 300 mm NaCl, 200 mm imidazole. The eluent was exchanged into 50 mm Tris-HCl (pH 7.5), 150 mm NaCl and concentrated. Thrombin cleavage of the affinity tag, when necessary, was performed using the Thrombin CleanCleave kit (Sigma) according to the manufacturer's instructions.Unlabeled or 6 tag was generated by in vitro reconstitution of the purified apoprotein  with heme, as follows. A 5-fold excess of hemin  was added into a 1 mm protein solution along with 2 mm disodium dithionite. The reactions were carried out in the dark and at room temperature under humidified argon gas for 16–18 h. Unbound heme was removed by incubating the reaction mixtures in the dark with equal volumes of 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 2 m imidazole for 16–18 h at 4 °C. Excess imidazole and unbound heme were removed by applying the reaction mixture to a desalting column (0.6 × 15 cm) packed with hydrated BIO-Gel P-6 DG resin (Bio-Rad).WT holo-CcmE bearing a C-terminal Hisin vitro heme reconstitution process. Whereas the presence of the His6 tag is important for efficient and timely covalent heme attachment, it causes significant protein loss at the cleavage step. Thus, a construct bearing a C-terminal His6 tag preceded by a thrombin cleavage site and followed by a Strep-II tag was used . The apoprotein was produced as described above and purified through its C-terminal His6 tag. Subsequently, covalent heme attachment and thrombin cleavage were performed as described above. After the cleavage reaction, the cleaved affinity tag was removed by applying the reaction mixture onto 5 ml of Strep-Tactin Sepharose (IBA), equilibrated with 50 mm Tris-HCl (pH 7.5), 150 mm NaCl. The flow-through was collected, and the resin was further washed with the equilibration buffer. Although this step led to ∼60–70% protein loss, due to association of the heme of holo-CcmE with the His6 tag that was bound on the affinity column, a sufficient amount of cleaved protein for NMR spectroscopy was recovered.WT holo-CcmE without an affinity tag was generated through a multistep 6 tag was generated by in vitro reconstitution of the purified apoprotein  with heme, as follows. H130A-apo-CcmE cleaved of its C-terminal His6 tag was mixed with excess heme  and incubated at room temperature for ∼30 min. The excess heme and DMSO were subsequently removed by buffer exchange in a concentrating device using 50 mm Tris-HCl (pH 7.5), 150 mm NaCl.H130A-holo-CcmE devoid of a Hiset al. (Protein concentration was determined using the Pierce BCA Reducing Agent Compatible Protein Assay Kit (Thermo Fisher Scientific). All purified proteins were subjected to SDS-PAGE analysis and electrospray ionization MS (ESI-MS) to confirm that they were pure and of the expected masses. SDS-PAGE analysis was carried out on 10% BisTris NuPAGE gels (Thermo Fisher Scientific) with prestained molecular weight markers . All samples were denatured at 100 °C for 2 min. Simply Blue Safe stain (Thermo Fisher Scientific) was used for staining gels for total protein content. For detection of proteins with covalently bound heme, the method of Goodhew et al.  was usedBFig. S3; details for acquisition of spectra are given below). The backbone chemical shifts of WT apo-CcmE recorded by 2D 1H-15N HSQC spectroscopy are in agreement with the published chemical shifts used for structural determination of apo-CcmE . Sample reduction was performed by the addition of several grains of sodium dithionite. In addition, absorption spectra in the presence of hydroxide and pyridine in reducing heme conditions were obtained  according to the method of Bartsch , 120 mm NaCl, 0.1% (v/v) Tween 20). For the detection of total CcmE amounts in crude membrane fractions, rabbit antiserum raised against E. coli CcmE  (dilution 1:1000) was used, followed by mouse anti-rabbit alkaline phosphatase–conjugated antibody (Sigma) . For the detection of total CcmC amounts in crude membrane fractions, a penta-His horseradish peroxidase alkaline phosphatase–conjugated mAb (Sigma) (dilution 1:2000) was used. Development was carried out using a SigmaFast 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium tablet (Sigma) in both cases. At least three replicates of each experiment were conducted.Immunoblotting was carried out following SDS-PAGE analysis by transferring the proteins onto nitrocellulose . Blocking was performed with 5% (w/v) skimmed milk powder in TBS (50 m1H frequencies of 600 and 750 MHz. The spectrometers were equipped with Oxford Instruments magnets and home-built triple-resonance pulsed-field gradient probes (750 MHz) or with Bruker Avance consoles and TCI CryoProbes (600 and 750 MHz). NMR data were acquired using either GE/Omega software with pulse sequences written in-house or Topspin software and pulse sequences provided in the Topspin libraries from Bruker Biospin. NMR data were processed using NMRPipe , at pH values ranging from 5.5 to 7.5. All experiments were conducted at 25 °C.NMR experiments were carried out using spectrometers operating at  NMRPipe . Protein1H-15N HSQC spectra were recorded at 600 MHz with spectral widths of 7,575.76 and 1,901.14 Hz in the 1H and 15N dimensions, respectively, or at 750 MHz with spectral widths of 9,433.96 and 2,380.95 Hz in the 1H and 15N dimensions, respectively. HSQC data sets were collected with 128 and 1024 complex points in F1 (15N) and F2 (1H), respectively. The peak height ratios shown in 2D 1H}-15N heteronuclear NOE experiments were recorded with and without 1H saturation for 4 s at 750 MHz in an interleaved manner. The data sets were acquired using the same sweep widths and numbers of data points as for the HSQC experiments. The {1H}-15N NOE was calculated as the ratio of the peak intensities in the spectra recorded with and without 1H saturation. Peak heights and errors in the {1H}-15N NOE were determined as described above.The {E. coli CcmE have been reported previously at pH 7.2 in 50 mm sodium phosphate, 300 mm NaCl at 30 °C (BMRB accession number 6723) (6 tag) and H130A-apo-CcmE  at 25 °C at pH values ranging from 5.5 to 7.5. Assignments under our solution conditions were confirmed using 3D 15N-edited total correlation spectroscopy-HSQC and NOESY-HSQC data sets.Resonance assignments for er 6723) . These aE. coli CcmC or CcmE residues of interest, 2,753 bacterial and archaeal representative proteomes from the NCBI database were searched for homologs of E. coli CcmC or CcmE, respectively, using blastp 2.2.28+ (NCBI) . Hits were used as a blastp query against the E. coli MG1655 proteome , and only the ones giving CcmC or CcmE as best-hit results were retained. The sequences were aligned using MUSCLE version 3.8.31 (E. coli MG1655 CcmC or CcmE homolog using MUSCLE 3.8.31.To assess the conservation of + (NCBI)  (e valuen 3.8.31  and checn 3.8.31 , and rep49 and Arg104 of E. coli CcmC and CcmE, respectively, in other Gram-negative bacteria, the same search procedure was used using blastp with e value <1E−5, identity >40%, and coverage >80%. A total of 628 were retrieved. Sequences were aligned using MUSCLE 3.8.31 (49 and Arg104 in the E. coli MG1655 CcmC and CcmE homologs, respectively, were retrieved. The frequency of occurrence of each pair was subsequently calculated on the subset of sequences from α- and γ-proteobacteria to avoid confounding effects due to excessive divergence.To identify amino acids found in positions corresponding to GlnE 3.8.31 , and resS. J. F., C. R., and D. A. I. M. designed research; S. H. S., D. G., and C. R. performed research; D. G., S. J. F., C. R., and D. A. I. M. analyzed the data; J. L. C. and C. K provided materials; C. R. and D. A. I. M. wrote the paper with contribution from the other authors.


The TRAINED group had lower glycemia , glycated hemoglobin , and triglycerides (98 (75-122) vs. 139 (109-214), p < 0.01); equal total cholesterol (199 (175-230) vs. 194 (165-220)), LDL-cholesterol (108 (83-133) vs. 109 (98-136)), and non-HDL-cholesterol (54 (30-74) vs. 62 (26-80)); and also higher HDL-cholesterol (64 (52-77) vs. 52 (44-63), p < 0.01) and LDL-C/oxLDL ratio  compared to the SED group. Proinflammatory cytokines as IL-1β , IL-6 , and TNF-α  were lower in the TRAINED group than in the SED group. The TRAINED group had lower total peroxides  and oxidized LDL  and higher total antioxidant capacity  compared to the SED group. In conclusion, in TRAINED women, BMI was lower, plasma lipid profile was better, plasma oxidative stress was diminished, and there was less expression of proinflammatory interleukins than in SED, suggesting that combined aerobic-resistance exercise training may promote the protection against the complications of ischemic cardio- and cerebrovascular disease in elderly women.Cardiovascular benefits for the general population of combined aerobic-resistance exercise training are well-known, but the impact of this exercise training modality on the plasma lipid, inflammatory, and antioxidant status in elderly women that are exposed to a great risk of developing ischemic cardio- and cerebrovascular diseases has not been well investigated. So, we aimed to evaluate the plasma lipids, oxidative stress, and inflammatory cytokines in 27 elderly women  that were performing moderate intensity combined aerobic-resistance exercise training (3 times/week for at least 18 months) and in 27 sedentary elderly women , not submitted to exercise training for at least 5 yrs. Our results showed that BMI was lower in the TRAINED group than in the SED group (25.1 ± 3.2 vs. 28.7 ± 5.1,  The pace of population aging is being accelerated, and the WHO estimated that between 2015 and 2050 the proportion of the world population over 60 years will grow from 12% to 22% [β), IL-6, and tumor necrosis factor alpha (TNF-α) [During the aging process, it is common to observe increases of adipose tissue in the viscera and organs such as the liver and muscles . Overwei (TNF-α) , 3. In t (TNF-α) .·, LOO·, and LO·, respectively) that further oxidize other lipid and protein molecules through massive production of reactive carbonyl species [Alterations in the plasma metabolism and concentration of lipoproteins , redox i species . Moreove species .The beneficial effects in the elderly population of aerobic and of resistance exercise training on the plasma lipid metabolism, oxidative, and inflammatory processes have been documented in the literature . WhereasFifty-four volunteer elderly women, aged 60-80 years, were selected for the study. A flow diagram is shown in None of the subjects presented asthma, type-1 diabetes mellitus; neoplastic, renal, or liver diseases; dementia; thrombosis; or manifested cardiovascular disease. None was under statin or other lipid-lowering drugs. The participants responded to a Food Frequency Questionnaire in which the consumption of antioxidants was assessed. The study was in agreement with the Declaration of Helsinki and with the Ethical Standards defined by Harriss and Atkinson  and was The physical exercise protocol is a combination of aerobic and resistance exercises performed in moderate intensity . The comVolunteers from the TRAINED group performed their prescribed exercises during 60-75 minutes per session, 3 times a week, on nonconsecutive days, for at least 18 months. The same experienced instructor supervised all the volunteers.A description of combined physical exercise regime performed by the TRAINED group is described in Volunteers of the SED group, although independent and active, were not involved in any regular exercise program for at least five years, and they were oriented to maintain their normal routine during the study.μL were obtained after centrifugation  of blood samples and stored at -80°C.Blood sampling occurred at 8:00 AM after a 12 h fast. The TRAINED group performed its last exercise training session at least 24 h beforehand. Serum/plasma aliquots of 500 Plasma total cholesterol concentration was measured by the CHOD-PAP method using commercial kits , and the results were analyzed with an automated system . HDL-C and triglyceride concentrations were determined using commercial kits and an automated analysis system . LDL-C was estimated by the Friedewald formula . Plasma β, IL-6, and TNF-α) using Millipore® Multiplex Assays Using Luminex® . All analyses were performed in accordance with the manufacturer's instructions.Serum concentration of oxLDL was evaluated by ELISA, using the Indirect Enzyme Immunoassay kit , total antioxidant capacity (TAC) by a colorimetric commercial kit , total lipid peroxide content (LOOH) using the QuantiChrom™ Peroxide Assay Kit, a colorimetric commercial kit , and proinflammatory cytokines . The Mann-Whitney test was used to determine the differences in plasma lipids and glucose, glycated hemoglobin, oxLDL, total peroxides, TAC, proinflammatory cytokines , and the relation between plasma concentrations of LDL-C and oxLDL ((LDL − C/oxLDL) × 100). Spearman's rank correlation coefficient was employed to identify any correlation between oxLDL and BMI or LDL-C or TAC or total peroxides. The significance level was set to 5% (p < 0.05).All data were previously analyzed by the Shapiro-Wilk test used to evaluate the normal distribution and after by the Levene test used to evaluate the variance homogeneity. Student's Physical and clinical characteristics of the volunteers are presented in p = 0.002). p = 0.02) and glycated hemoglobin (p = 0.04) were lower in the TRAINED group than in the SED group. LDL-C and non-HDL-C were similar but HDL-C was higher (p = 0.001) and triglycerides were lower (p = 0.008) in the TRAINED group compared to the SED group.As shown in p < 0.001), whereas LOOH (p < 0.001) and oxLDL (p = 0.02) were lower in the TRAINED group than in the SED group. In addition, as shown in p = 0.04).As shown in β (p = 0.001), IL-6 (p = 0.02), and TNF-α (p = 0.01) was higher in the SED group than in the TRAINED group.Spearman's coefficient analysis  showed aIn this transversal study enrolling elderly women, it was shown that the regular practice of combined aerobic-resistance exercise leads to a clear-cut improvement of markers of lipid and inflammatory status and of the oxidative stress that are related to the prevention of the manifestations of atherosclerosis.LDL-C was not lower in the TRAINED group than in the SED group, which is in accordance with studies showing that several modalities of exercise training do not alter LDL-C plasma levels , 18. HowBesides the decrease in the plasma levels of oxLDL, an established risk factor for ischemic cardio- and cerebrovascular diseases –26, we aHere, the TRAINED group exhibited higher levels of HDL-C as compared to the SED group. Exercise training is a classical factor to promote an increase in HDL-C , 36–38, As judged from the comparison of the TRAINED and SED groups, the practice of combined aerobic-resistance exercise training also had anti-inflammatory actions, since the TRAINED group presented lower serum levels of proinflammatory cytokines than the SED group. Similar to our results, some authors have reported that combined aerobic-resistance exercise training improves the inflammatory status –46. It iIt is also widely accepted that exercise training is a chief factor to attain the control of glycemic levels, which was also documented here and in other studies , 49, 50.As a limitation of this study, it should be mentioned that a longitudinal interventional study would be rigorously more appropriate to establish the biohumoral changes elicited by exercise training. However, in this cross-sectional protocol, the results of long-standing (over 18 months) regular practice of training, as compared to nonpractitioners of the same community, offer a fair evaluation of individuals in their real-life conditions.The results of the current cross-sectional study suggest that elderly women may benefit from the regular practice of combined aerobic-resistance exercise training in many metabolic aspects that are related to protection against the complications of ischemic cardio- and cerebrovascular disease.